Macrophage Iron Retention Research Articles

Comparative analysis of the functional properties of human and mouse ferroportin.

Ferroportin (Fpn)-expressed at the plasma membrane of macrophages, enterocytes, and hepatocytes-mediates the transfer of cellular iron into the blood plasma. Under the control of the iron-regulatory hormone hepcidin, Fpn serves a critical role in systemic iron homeostasis. Although we have previously characterized human Fpn, a great deal of research in iron homeostasis and disorders uses mouse models. By way of example, the flatiron mouse, a model of classical ferroportin disease, bears the mutation H32R in Fpn and is characterized by systemic iron deficiency and macrophage iron retention. The flatiron mouse also appears to exhibit a manganese phenotype, raising the possibility that mouse Fpn serves a role in manganese metabolism. At odds with this observation, we have found that human Fpn does not transport manganese, so we considered the possibility that a species difference could explain this discrepancy. We tested the hypothesis that mouse but not human Fpn can transport manganese and performed a comparative analysis of mouse and human Fpn. We examined the functional properties of human Fpn, mouse Fpn, and mutant mouse Fpn by using radiotracer assays in RNA-injected Xenopus oocytes. We found that neither mouse nor human Fpn transports manganese. Mouse and human Fpn share identical properties with respect to substrate profile, calcium dependence, optimal pH, and hepcidin sensitivity. We have also demonstrated that Fpn is not an ATPase pump. Our findings validate the use of mouse models of ferroportin function in iron homeostasis and disease.

Tissue iron distribution in patients with anemia of inflammation: Results of a pilot study.

Anemia of inflammation (AI) is frequently present in subjects with inflammatory disorders, primarily caused by inflammation-driven iron retention in macrophages. So far, only limited data on qualitative and quantitative estimates of tissue iron retention in AI patients exist. We performed a prospective cohort study analyzing splenic, hepatic, pancreatic, and cardiac iron content with MRI-based R2*-relaxometry in AI patients, including subjects with concomitant true iron deficiency (AI+IDA) hospitalized between 05/2020-01/2022. Control groups were individuals without inflammation. Spleen R2* values in AI patients with ferritin ≤200 μg/L (AI+IDA) were comparable with those found in controls. In AI patients with ferritin >200 μg/L, spleen (47.6s-1 vs. 19.3 s-1 , p < .001) and pancreatic R2* values (32.5s-1 vs. 24.9 s-1 , p=.011) were significantly higher compared with controls, while liver and heart R2*-values did not differ. Higher spleen R2* values were associated with higher ferritin, hepcidin, CRP, and IL-6 concentrations. Spleen R2* values normalized in AI patients after recovery (23.6s-1 vs. 47.6 s-1 , p=.008), while no changes were found in patients with baseline AI+IDA. This is the first study investigating tissue iron distribution in patients with inflammatory anemia and AI with concomitant true iron deficiency. The results support the findings in animal models demonstrating iron retention in macrophages, which are primarily accumulating in the spleen under inflammatory conditions. MRI-related iron measurement may help to better characterize actual iron needs and to define better biomarker thresholds in the diagnosis of true ID in patients with AI. It may qualify as a useful diagnostic method to estimate the need for iron supplementation and to guide therapy.

A study of an Incidence of tuberculosis is linked to elevated hepcidin at HIV diagnosis

Background: Hepcidin prevents ferroportin-mediated iron efflux, which results in intracellular macrophage iron retention and may encourage Mycobacterium tuberculosis nutrient absorption and TB pathogenesis. Aims &amp; Objectives: In order to research into the relationship between incident pulmonary and extrapulmonary TB and plasma hepcidin levels, a retrospective cohort of HIV-positive, antiretroviral-native individuals had their plasma hepcidin levels tested at HIV diagnosis. Methods &amp; Materials: Between 5 August 2017 and 1 June 2019, 140 individuals were monitored, and 34 incident TB cases were found. Discussion: After a median time to TB of 7 months, higher hepcidin was linked to a significantly increased chance of TB. Conclusion: The need for time-sensitive biomarkers that can detect individual variations in TB risk and the elucidation of iron-related causative pathways cannot be overstated.

Iron status in chronic inflammatory disease: therapeutic implications.

Anemia of inflammation (AI) is a very frequent clinical condition affecting globally more than a billion people with chronic inflammatory disorders, such as chronic kidney disease, heart failure, and inflammatory bowel disease. It is usually associated with iron deficiency (ID), which imposes a severe additional burden on the recovery from the primary disease. The pathophysiology of iron dysregulation that may ultimately lead to absolute iron deficiency anemia (IDA) during inflammation is multifactorial and includes reduced iron absorption in the bowel, iron retention in macrophages of the reticuloendothelial system, reduction in circulatory half‑life of erythrocytes, inadequate production and activity of erythropoietin, and impaired proliferation and differentiation of erythroid progenitor cells. These result in hypoferremia and iron-restricted erythropoiesis. AI is mostly mild to moderate, normochromic and normocytic with normal or even increased ferritin levels. The current treatment options for AI include iron replacement therapy, treatment with erythropoiesis‑stimulating agents, and red blood cell transfusion. ID management is based on oral or intravenous iron preparations. Given the pathophysiology, treatment with oral iron, although widely used, presents several limitations that impact its effectiveness in patients with chronic inflammatory conditions. Instead, intravenous iron preparations are a valuable option for patients with chronic inflammatory diseases, as they overcome reduced bowel absorption. Novel therapeutic approaches include downregulation of hepcidin synthesis and function, and stabilization of the hypoxia‑inducible factor via inhibition of prolyl hydroxylase domain. Several studies in vitro and in vivo are ongoing; however, the results in humans are still elusive.

Pharmacological Targeting of BMP6-SMAD Mediated Hepcidin Expression Does Not Improve the Outcome of Systemic Infections With Intra-Or Extracellular Gram-Negative Bacteria in Mice

IntroductionHepcidin is the systemic master regulator of iron metabolism as it degrades the cellular iron exporter ferroportin. In bacterial infections, hepcidin is upregulated to limit circulating iron for pathogens, thereby increasing iron retention in macrophages. This mechanism withholds iron from extracellular bacteria but could be of disadvantage in infections with intracellular bacteria. We aimed to understand the role of hepcidin in infections with intra- or extracellular bacteria using different hepcidin inhibitors.MethodsFor the experiments LDN-193189 and oversulfated heparins were used, which interact with the BMP6-SMAD pathway thereby inhibiting hepcidin expression. We infected male C57BL/6N mice with either the intracellular bacterium Salmonella Typhimurium or the extracellular bacterium Escherichia coli and treated these mice with the different hepcidin inhibitors.ResultsBoth inhibitors effectively reduced hepcidin levels in vitro under steady state conditions and upon stimulation with the inflammatory signals interleukin-6 or lipopolysaccharide. The inhibitors also reduced hepcidin levels and increased circulating iron concentration in uninfected mice. However, both compounds failed to decrease liver- and circulating hepcidin levels in infected mice and did not affect ferroportin expression in the spleen or impact on serum iron levels. Accordingly, both BMP-SMAD signaling inhibitors did not influence bacterial numbers in different organs in the course of E.coli or S.Tm sepsis.ConclusionThese data indicate that targeting the BMP receptor or the BMP-SMAD pathway is not sufficient to suppress hepcidin expression in the course of infection with both intra- or extracellular bacteria. This suggests that upon pharmacological inhibition of the central SMAD-BMP pathways during infection, other signaling cascades are compensatorily induced to ensure sufficient hepcidin formation and iron restriction to circulating microbes.

Macrophage iron retention aggravates atherosclerosis: Evidence for the role of autocrine formation of hepcidin in plaque macrophages

Iron accumulation has been frequently found in atherosclerotic lesions, especially in macrophages/foam cells, but the exact mechanisms by which hepcidin induces iron retention in plaque macrophages and its roles in atherogenesis remain unknown. Double immunofluorescence staining showed colocalization of hepcidin-positive macrophages with ox-LDL, TLR4, p-p65 and ferritin light chain (ferritin-L) both in human and murine atherosclerotic lesions. RAW264.7 macrophages incubated with ox-LDL showed elevated expression of TLR4, p-p65, hepcidin, ferritin-L/H, CYP27A1, CD36, PPARγ, liver X receptor α (LXRα), and ATP binding cassette transporter A1/G1 (ABCA1/G1), as well as increased intracellular labile iron pool level and lipid accumulation. Ox-LDL-induced iron retention and lipid accumulation were aggravated by lipopolysaccharide but blocked by TAK-242, an antagonist of TLR4. Moreover, macrophage TLR4/NF-κB pathway activation and foaming triggered by ox-LDL was enhanced by ferric ammonium citrate or exogenous hepcidin but attenuated by hepcidin silencing or the use of iron chelator. Meanwhile, the addition of hepcidin stimulated CD36-mediated Dil-labeled-ox-LDL uptake and inhibited the LXRα-ABCA1/G1 pathway-dependent cholesterol efflux in macrophages, which was significantly reversed by 27-hydroxycholesterol but further exacerbated by cyclosporin A, a selective inhibitor of CYP27A1. Our study provided the evidence that iron trapped in atherosclerosis plaque macrophages contributes to cholesterol disequilibrium-initiated foam cell formation, which is provoked by the unique but largely unknown autocrine formation of hepcidin in plaque macrophages via activating the TLR4/NF-κB pathway when exposed to ox-LDL. Such findings, considering the intricate vicious cycle between macrophage hepcidin autocrine-triggered iron retention and cholesterol disequilibrium, may shed new light on the “iron hypothesis” of atherosclerosis.

Established and Emerging Concepts to Treat Imbalances of Iron Homeostasis in Inflammatory Diseases.

Inflammation, being a hallmark of many chronic diseases, including cancer, inflammatory bowel disease, rheumatoid arthritis, and chronic kidney disease, negatively affects iron homeostasis, leading to iron retention in macrophages of the mononuclear phagocyte system. Functional iron deficiency is the consequence, leading to anemia of inflammation (AI). Iron deficiency, regardless of anemia, has a detrimental impact on quality of life so that treatment is warranted. Therapeutic strategies include (1) resolution of the underlying disease, (2) iron supplementation, and (3) iron redistribution strategies. Deeper insights into the pathophysiology of AI has led to the development of new therapeutics targeting inflammatory cytokines and the introduction of new iron formulations. Moreover, the discovery that the hormone, hepcidin, plays a key regulatory role in AI has stimulated the development of several therapeutic approaches targeting the function of this peptide. Hence, inflammation-driven hepcidin elevation causes iron retention in cells and tissues. Besides pathophysiological concepts and diagnostic approaches for AI, this review discusses current guidelines for iron replacement therapies with special emphasis on benefits, limitations, and unresolved questions concerning oral versus parenteral iron supplementation in chronic inflammatory diseases. Furthermore, the review explores how therapies aiming at curing the disease underlying AI can also affect anemia and discusses emerging hepcidin antagonizing drugs, which are currently under preclinical or clinical investigation.

Macrophage ferroportin is essential for stromal cell proliferation in wound healing.

Iron recycling by macrophages is essential for erythropoiesis, but may also be relevant for iron redistribution to neighboring cells at the local tissue level. Using mice with iron retention in macrophages due to targeted inactivation of the iron exporter ferroportin, we investigated the role of macrophage iron release in hair follicle cycling and wound healing, a complex process leading to major clinical problems, if impaired. Genetic deletion of ferroportin in macrophages resulted in iron deficiency and decreased proliferation in epithelial cells, which consequently impaired hair follicle growth and caused transient alopecia. Hair loss was not related to systemic iron deficiency or anemia, thus indicating the necessity of local iron release from macrophages. Inactivation of macrophage ferroportin also led to delayed skin wound healing with defective granulation tissue formation and diminished fibroplasia. Iron retention in macrophages had no impact on the inflammatory processes accompanying wound healing, but affected stromal cell proliferation, blood and lymphatic vessel formation, and fibrogenesis. Our findings reveal that iron/ferroportin plays a largely underestimated role in macrophage trophic function in skin homeostasis and repair.

Anti-TNF-Mediated Modulation of Prohepcidin Improves Iron Availability in Inflammatory Bowel Disease, in an IL-6-Mediated Fashion.

Background. Anaemia is common in inflammatory bowel disease (IBD), frequently resulting from a combination of iron deficiency and of anaemia of chronic disease (ACD). ACD is characterized by macrophage iron retention induced by proinflammatory cytokines. Hepcidin is the master inducer of iron accumulation during ACD, and its production is mainly regulated by IL-6 and the novel erythroid hormone erythroferrone (ERFE). This study evaluates whether anti-TNF monoclonal antibodies therapy modurates hepcidin production and the levels of its main regulators, leading to a restoration of iron homeostasis. Methods. Sera were collected from 21 IBD patients, before each anti-TNF administration, for the first 6 weeks of therapy. Prohepcidin, erythropoietin, erythroferrone, C reactive protein, interleukin-6, iron markers, and haemoglobin levels were measured and clinical activity indexes were evaluated. Results. Serum prohepcidin, IL-6, CRP, and ferritin were significantly reduced after 6-week treatment; an increase in serum iron and total transferrin was observed. No changes in the EPO-ERFE axis were found. Remarkably, haemoglobin was significantly increased. Conclusions. Anti-TNF therapy improves iron metabolism and, subsequently, anaemia in IBD. This effect appears to be related to the modulation of the cytokine network and specifically IL-6 leading to a relevant decrease of hepcidin, a master regulator of ACD.

Iron gene expression profile in atherogenic Mox macrophages

RationaleThe role of macrophage iron in the physiopathology of atherosclerosis is an open question that needs to be clarified. In atherosclerotic lesions, recruited macrophages are submitted to cytokines and oxidized lipids which influence their phenotype. An important phenotypic population driven by oxidized phospholipids is the Mox macrophages which present unique biological properties but their iron phenotype is not well described. ObjectiveTo investigate the effect of Mox polarization by oxidized LDL (oxLDL) on macrophage iron metabolism in the absence or presence of proinflammatory stimuli. MethodsBone marrow-derived macrophages were treated with different sources of LDL and/or LPS/IFNγ (M1 activator). Expression of ferroportin (Slc40a1, alias Fpn), heme oxygenase-1 (Hmox1), H- and L-ferritin (Fth1 and Ftl1), hepcidin (Hamp), ceruloplasmin (Cp) and interleukine-6 (Il6) was followed by quantitative PCR. FPN and HMOX1 protein expression was analyzed by immunofluorescence and in-cell-Western blotting. ResultsMox macrophages expressed increased Hmox1 and Fth1 levels with basal FPN protein levels despite the significant increase of Fpn mRNA. Upregulation of Hmox1 and Fpn mRNA was specific to LDL oxidative modification and mediated by NRF2. The downregulation of both Cp isoforms and the upregulation of Hamp expression observed in Mox macrophages suggest that FPN mediated iron export could be compromised. Simultaneous exposure to oxLDL and LPS/IFNγ leads to a mixed Mox/M1 phenotype that is closer to M1. ConclusionA microenvironment rich in oxLDL and proinflammatory cytokines could promote macrophage iron retention and lipid accumulation profiles, a specific cell phenotype that likely contributes to lesion development and plaque instability in atherosclerosis.

Rapidly Escalating Hepcidin and Associated Serum Iron Starvation Are Features of the Acute Response to Typhoid Infection in Humans.

BackgroundIron is a key pathogenic determinant of many infectious diseases. Hepcidin, the hormone responsible for governing systemic iron homeostasis, is widely hypothesized to represent a key component of nutritional immunity through regulating the accessibility of iron to invading microorganisms during infection. However, the deployment of hepcidin in human bacterial infections remains poorly characterized. Typhoid fever is a globally significant, human-restricted bacterial infection, but understanding of its pathogenesis, especially during the critical early phases, likewise is poorly understood. Here, we investigate alterations in hepcidin and iron/inflammatory indices following experimental human typhoid challenge.Methodology/Principal FindingsFifty study participants were challenged with Salmonella enterica serovar Typhi and monitored for evidence of typhoid fever. Serum hepcidin, ferritin, serum iron parameters, C-reactive protein (CRP), and plasma IL-6 and TNF-alpha concentrations were measured during the 14 days following challenge. We found that hepcidin concentrations were markedly higher during acute typhoid infection than at baseline. Hepcidin elevations mirrored the kinetics of fever, and were accompanied by profound hypoferremia, increased CRP and ferritin, despite only modest elevations in IL-6 and TNF-alpha in some individuals. During inflammation, the extent of hepcidin upregulation associated with the degree of hypoferremia.Conclusions/SignificanceWe demonstrate that strong hepcidin upregulation and hypoferremia, coincident with fever and systemic inflammation, are hallmarks of the early innate response to acute typhoid infection. We hypothesize that hepcidin-mediated iron redistribution into macrophages may contribute to S. Typhi pathogenesis by increasing iron availability for macrophage-tropic bacteria, and that targeting macrophage iron retention may represent a strategy for limiting infections with macrophage-tropic pathogens such as S. Typhi.

Elevated hepcidin at HIV diagnosis is associated with incident tuberculosis in a retrospective cohort study.

Hepcidin inhibits ferroportin-mediated iron efflux, leading to intracellular macrophage iron retention, possibly favoring Mycobacterium tuberculosis iron acquisition and tuberculosis (TB) pathogenesis. Plasma hepcidin was measured at human immunodeficiency virus (HIV) diagnosis in a retrospective HIV-prevalent, antiretroviral-naïve African cohort to investigate the association with incident pulmonary and/or extra-pulmonary TB. One hundred ninety-six participants were followed between 5 August 1992 and 1 June 2002, with 32 incident TB cases identified. Greater hepcidin was associated with significantly increased likelihood of TB after a median time to TB of 6 months. Elucidation of iron-related causal mechanisms and time-sensitive biomarkers that identify individual changes in TB risk are needed.

Inflammation and ER stress downregulate BDH2 expression and dysregulate intracellular iron in macrophages.

Macrophages play a very important role in host defense and in iron homeostasis by engulfing senescent red blood cells and recycling iron. Hepcidin is the master iron regulating hormone that limits dietary iron absorption from the gut and limits iron egress from macrophages. Upon infection macrophages retain iron to limit its bioavailability which limits bacterial growth. Recently, a short chain butyrate dehydrogenase type 2 (BDH2) protein was reported to contain an iron responsive element and to mediate cellular iron trafficking by catalyzing the synthesis of the mammalian siderophore that binds labile iron; therefore, BDH2 plays a crucial role in intracellular iron homeostasis. However, BDH2 expression and regulation in macrophages have not yet been described. Here we show that LPS-induced inflammation combined with ER stress led to massive BDH2 downregulation, increased the expression of ER stress markers, upregulated hepcidin expression, downregulated ferroportin expression, caused iron retention in macrophages, and dysregulated cytokine release from macrophages. We also show that ER stress combined with inflammation synergistically upregulated the expression of the iron carrier protein NGAL and the stress-inducible heme degrading enzyme heme oxygenase-1 (HO-1) leading to iron liberation. This is the first report to show that inflammation and ER stress downregulate the expression of BDH2 in human THP-1 macrophages.

Erythropoiesis and Iron Metabolism In Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red cell aplasia characterized by macrocytic anemia, reticulocytopenia and reduced number of erythroblasts in the bone marrow. Etiology and symptoms of DBA are closely associated with mutations of genes coding for 12 ribosomal proteins. Approximately 40% of patients are dependent on regular transfusions. In chronically transfused DBA patients, severe iron overload with tissue hemosiderosis with the need for iron chelation develops and has a substantial impact on morbidity and mortality of DBA patients. Liver is the most affected organ, but extra-hepatic iron accumulation, although less well documented, appears to occur early and at high frequency. Despite these facts a detailed analysis of iron metabolism in DBA is missing.We therefore aimed to determine selected markers of erythropoid activity and iron metabolism including the levels of the key regulator of iron homeostasis, hepcidin, in 14 DBA patients from the Czech National DBA Registry. Ten patients receive regular transfusions, two are treated with steroids and two are in the remission. None of the patients had signs of an inflammatory process at the time of the examination. Following markers of iron metabolism were analysed: iron, total iron binding capacity, transferrin saturation, ferritin and soluble transferrin receptor (TfR). Serum hepcidin was measured by proteomic analysis. Levels of rowth differentiation factor 15 (GDF 15), aputative marker of erythroid activity, were measured by ELISA. Pearls' staining was used for the liver iron detection. In all transfusion dependent patients, low levels of soluble TfR and reduced number of erythroblasts in the bone marrow confirm severally suppressed erythropoiesis which corresponded with dramatically elevated serum erythropoietin (EPO) levels. Analysis of iron parameters showed increased transferrin saturation and high ferritin levels. The two patients who are currently in the remission had near normal blood counts and iron parameters. Liver biopsy on 5 selected patients revealed excessive iron accumulation in both liver hepatocytes and macrophages which can be attributed to increased iron uptake and non-effective erythrocytes-derived iron recycling, respectively. Measurement of hepcidin levels showed significant elevation in DBA cohort in comparison with healthy controls (p>0.0005). The highest hepcidin was detected for transfusion dependent patients, the lowest (near normal) for patients in remission. As erythropoiesis is known to produce a signal for hepcidin suppression, patients in remission with restored erythropoietic activity are likely able to attenuate hepcidin expression and increase the pool of iron available for improved erythropoiesis. On the other hand repeated blood transfusions contribute to the suppression of erythropoiesis thus likely relieving hepcidin synthesis and worsening iron recycling. We also found that elevated hepcidin positively correlated with ferritin levels. No correlation was detected with EPO or GDF15, the putative negative regulator of hepcidin production. These results indicate that EPO is an indirect suppressor of hepcidin synthesis and that GDF15 possibly plays a hepcidin-regulatory role in the disease states associated with ineffective erythropoiesis but not in DBA.In conclusion we propose that in DBA patients iron overload develops as a consequence of the combination of repeated transfusions and absent or reduced erythropoiesis in the bone marrow. At the systemic level the increased hepcidin promotes iron retention in macrophages and thus is responsible for impaired iron recycling. AcknowledgmentsGrant support: Ministry of Health, Czech Republic, grant No. NT11059 to DP and NT/13587 to VD, Czech Grant Agency, grant No. P305/11/1745, and Internal Grant of Palacky University Olomouc (LF_2013_010). We thank dr. Thomas Ganz for validation of our HPLC-MS-based hepcidin measurements. Disclosures:No relevant conflicts of interest to declare.

The Role of Prohepcidin in Anemia Due to Helicobacter pylori Infection

Background: Hepcidin, a key regulator of iron homeostasis, increases when inflammation and some infections occur. It plays a critical role in macrophage iron retention, which underlies inflammation/infection caused anemia. It is known that Helicobacter pylori (HP) may lead to iron deficiency (ID) due to occult blood loss or reduced iron absorption. This study investigates the role of prohepcidin, hepcidin's precursor, in ID and ID anemia (IDA) with a concurrent HP infection. Methods: In this prospectively designed study, 15 patients with IDA and a concurrent HP infection (group 1), 11 patients with an ID and a concurrent HP infection (group 2), and 18 patients with HP infection (group 3) were observed. All groups received only HP eradication therapy. Twenty-five age- and sex-matched children without ID/IDA and HP infection were included in the study as the control group. In all groups and control group, measurements were taken for pre- and posttreatment hemoglobin, serum prohepcidin, serum ferritin, serum iron (SI), transferrin saturation, erythrocyte sedimentation rate, fibrinogen, and C-reactive protein levels. Results: The pretreatment prohepcidin levels were significantly higher only in group 1 compared to the control group (P < .05). In group 1, a significant increase in hemoglobin and SI levels and a significant reduction in prohepcidin levels were additionally observed following HP eradication treatment (P < .05). However, in groups 2 and 3, significant differences in hemoglobin, iron, and prohepcidin levels between pre- and posttreatment were not observed. Conclusion: Elevated serum prohepcidin might indicate the role of inflammation in the etiology of anemia concurrent with HP.

Rescuing iron‐overloaded macrophages by conservative relocation of the accumulated metal

Systemic iron deficiency concomitant with macrophage iron retention is characteristic of iron-refractory anaemias associated with chronic disease. The systemic misdistribution of iron, which is further exacerbated by parenteral iron supplementation, is mainly attributable to iron retention exerted on resident macrophages by hepcidin-mediated down-regulation of the iron exporter ferroportin. We aimed at developing an experimental macrophage-based cell model that recapitulates pathophysiological features of iron misdistribution found in chronic disorders and use it as a screening platform for identifying agents with the potential for relocating the accumulated metal and restoring affected functions. A raw macrophage subline was selected as cell model of iron retention based on their capacity to take up polymeric iron or aged erythrocytes excessively, resulting in a demonstrable increase of cell labile iron pools and oxidative damage that are aggravated by hepcidin. This model provided a three-stage high throughput screening platform for identifying agents with the combined ability to: (i) scavenge cell iron and thereby rescue macrophage cells damaged by iron-overload; (ii) bypass the ferroportin blockade by conveying the scavenged iron to other iron-starved cells in co-culture via transferrin but (iii) without promoting utilization of the scavenged iron by intracellular pathogens. As test agents we used chelators in clinical practice and found the oral chelator deferiprone fulfilled essentially all of the three criteria. We provide a proof of principle for conservative iron relocation as complementary therapeutic approach for correcting the misdistribution of iron associated with chronic disease and exacerbated by parenteral iron supplementation.

Elevated Serum Hepcidin in Patients with AML Prior to and After Allogeneic Hematopoietic Cell Transplantation Does Not Correlate with Transfusional Body Iron, HFE Genotype or Graft Versus Host Disease and May Protect From Excessive Parenchymal Iron Loading.

Abstract 4047Poster Board III-982Hepcidin (hep), a 25-amino-acid peptide, is the central regulator of iron homeostasis. Its transcription is upregulated by inflammatory cytokines and iron and is downregulated by iron deficiency, ineffective erythropoiesis, and hypoxia. Also HFE gene mutations are associated with less liver hepcidin messenger RNA. Both inherited (HFE genotype) and treatment-related factors influencing hep expression in patients (pts) with AML prior to and after allogeneic hematopoietic cell transplantation (HCT) as blood transfusions (BT), body iron and anemia were studied. The impact of chemotherapy, conditioning regimen, and Graft versus Host Disease (GvHD) on serum hep was analysed. Patients and methods42 consecutive pts (23 male/19 female, median age 57 [range:18-70] years) with AML who underwent allogeneic HCT from February, 2008 - February, 2009 at the University of Leipzig were included. Each patient was assessed 10 days prior to and at a median of 3 (range: 3-5) months after HCT. Donors were matched related in 8 (19%) and matched unrelated (MUD) in 34 (81%) pts. Preparative regimen consisted of 12 Gy TBI/cyclophosphamid 120 mg/kg (ATG was included for unrelated HCT) in 13 (31%) and fludarabin 30 mg/m2/day for 3 days/2 Gy TBI) in 29 (69%) pts. Acute GvHD > grade II was present in 13 (31%) and chronic GvHD in 17 (40%) pts. HFE genotype prior to and after HCT was assessed by PCR technique. Body iron was assessed by serum ferritin (sf) (normal values < 400ng/mL). Serum hep was measured by hepcidin C-ELISA at Intrinsic LifeSciences LLC, La Jolla, CA.(normal values: male 29-254 ng/mL, female: 17-286 ng/mL). Hep levels of 21 age-and gender-matched healthy volunteers (6 m/15 f, median age 57 years) were used as a control. ResultsMedian serum hep was much higher in pts both prior to [median 358 (range:56-1096) ng/ml] and after HCT [median 398 (range:172-941) ng/ml] compared with the control group [median 52 (range:8.3-131) ng/ml] (p<0.0001). Age and gender had no influence on hep values. Similarly, liver function, interval between diagnosis and HCT, number of chemotherapies, conditioning regimen, antibiotic- or antifungal-treatments had no impact on hep level. Iron overload was already seen in all pts prior to HCT with a median sf of 1945 (range: 617-6981) ng/mL after a median number of 22 units BT. Although after HCT the number of BT mounted to a median of 30 units (p<0.0001), sf with a median of 2260 ng/mL remained elevated comparable to the level prior to HCT. Lower hep levels significantly correlated with fewer BT (p=0.001), but surprisingly not with sf values. Hep correlated inversely with the degree of anemia (p=0.002). Mutations in the HFE gene were found in 19 (46%) pts prior to HCT (heterozygosity (het) for H63D, n=11, het C282Y, n=3, het S65C, n=1, and homozygosity (homo) for H63D, n=4) and in 15 (37.5%) pts after HCT reflecting donor genotype (het for H63D,n=12, het C282Y, n=1, compound-het, n=1). Mutations in the HFE gene were not associated with lower hep levels.After HCT, 19 (45%) pts showed a decline in hep level of 155 (range: 394.8-9.5) ng/ml and 23 (55%) pts had an increase in hep levels of 138 (range: 43.3- 620.9) ng/ml compared with pre-transplantaion levels. None of the above mentioned parameters could predict or correlate with these changes in serum hep. Iron overload prior to HCT strongly correlated with later extensive chronic GvHD (p=0.003) and tended to correlate with limited GvHD (p=0.06). On the other hand, hep levels at any time point did not correlate with acute or later chronic GvHD. ConclusionsSerum hepcidin is highly elevated in pts with AML prior to as well as after allogeneic HCT compared with healthy controls mainly because of frequent blood transfusions leading to elevated iron stores. This suggests that hepcidin synthesis and upregulation remain intact despite intensive chemotherapy and HCT. Hepcidin normally binds to ferroportin, leading to intracellular retention of iron in macrophages and to a reduction of extracellular serum iron. This may explain why serum hepcidin correlates with blood transfusions but not with serum ferritin values. Actually, overexpression of hepcidin may play an important protective role in this setting as it may prevent an increased ferroportin-mediated iron export from macrophages thereby reducing the severity of parenchymal iron loading and damage. Disclosures:Westerman:INTRINSIC LIFESCIENCES LLC: Consultancy, Employment, Equity Ownership. Hehme:Novartis: Employment. Niederwieser:Novartis: Speakers Bureau. Al-Ali:Novartis: Research Funding.

Regulation of iron homeostasis in anemia of chronic disease and iron deficiency anemia: diagnostic and therapeutic implications

AbstractThe anemia of chronic disease (ACD) is characterized by macrophage iron retention induced by cytokines and the master regulator hepcidin. Hepcidin controls cellular iron efflux on binding to the iron export protein ferroportin. Many patients, however, present with both ACD and iron deficiency anemia (ACD/IDA), the latter resulting from chronic blood loss. We used a rat model of ACD resulting from chronic arthritis and mimicked ACD/IDA by additional phlebotomy to define differing iron-regulatory pathways. Iron retention during inflammation occurs in macrophages and the spleen, but not in the liver. In rats and humans with ACD, serum hepcidin concentrations are elevated, which is paralleled by reduced duodenal and macrophage expression of ferroportin. Individuals with ACD/IDA have significantly lower hepcidin levels than ACD subjects, and ACD/IDA persons, in contrast to ACD subjects, were able to absorb dietary iron from the gut and to mobilize iron from macrophages. Circulating hepcidin levels affect iron traffic in ACD and ACD/IDA and are more responsive to the erythropoietic demands for iron than to inflammation. Hepcidin determination may aid to differentiate between ACD and ACD/IDA and in selecting appropriate therapy for these patients.

Effects of the Aspergillus fumigatus siderophore systems on the regulation of macrophage immune effector pathways and iron homeostasis

The saprophytic fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen, which is responsible for invasive aspergillosis in immunocompromised patients. Iron plays an essential role for the growth and proliferation of A. fumigatus. This fungus synthesizes three major siderophores. It excretes triacetylfusarinine C to capture iron, while it accumulates ferricrocin and hydroxyferricrocin for hyphal and conidial iron storage, respectively. Herein, we investigated the role of the siderophore system of A. fumigatus in the modulation of immune effector pathways and iron homeostasis in macrophages. We set up a co-culture system consisting of the murine macrophage cell line RAW264.7 and either A. fumigatus wild type or a siderophore-deficient mutant (Δ sidA). We used real-time quantitative RT-PCR and Western blot analyses to study the expression of macrophage iron metabolism and innate immune response genes in response to pathogen challenge. Infection of macrophages with A. fumigatus wild type, but not with the Δ sidA mutant, induced expression of TNF and phagocyte oxidase subunit 47 at the transcriptional level. Moreover, infection with A. fumigatus wild type, but not with the Δ sidA mutant, compromised macrophage iron homeostasis. Infection with wild-type A. fumigatus decreased expression of the two cellular iron importers, the divalent metal transporter-1 and the transferrin receptor, and the only known iron exporter ferroportin. At the same time, it increased macrophage iron retention and ferritin synthesis. These data indicate that A. fumigatus affects the regulation of macrophage iron homeostasis and innate immune effector pathways via its siderophore system. The changes in immune response may be a consequence of macrophage iron restriction.

Hepcidin and iron-related gene expression in subjects with Dysmetabolic Hepatic Iron Overload

Many patients with hepatic iron overload do not have identifiable mutations and often present with metabolic disorders and hepatic steatosis. Since the pathophysiology of Dysmetabolic Hepatic Iron Overload (DHIO) is still obscure, the aim of this study was to evaluate, in these patients, possible alterations in iron-related molecule expression. Iron-related gene mRNA levels were determined by quantitative-PCR in liver biopsies of subjects with NAFLD without iron overload and patients with HFE-hemochromatosis, beta-thalassemia major and DHIO. Urinary hepcidin was measured by immunoblotting. No alterations in mRNA expression of either iron transporters or exporters were found in DHIO. mRNA and urinary hepcidin levels normalized for the amount of iron overload showed a significantly lower ratio than in controls, although not as low as in hemochromatosis or beta-thalassemia. Differently from what observed in hemochromatosis, hepcidin mRNA did not correlate with urinary hepcidin. Patients with DHIO show appropriate regulation of mRNAs encoding proteins involved in iron uptake and efflux but dysregulation of hepcidin production. The relatively elevated urinary hepcidin can explain the iron phenotype in DHIO (more macrophage iron retention and low/normal transferrin saturation).